Anthrax a zoonotic disease is caused by Gram positive, sporulating bacteria, Bacillus anthracis. Protective antigen, PA is the major component of all the vaccines against anthrax. Till date, culture supernatants of B. anthracis have been the major source of purifying PA. However, working with B. anthracis cultures requires P3 facilities that are cost-prohibitive. Apart from this, PA preparation from B. anthracis is often contaminated with other anthrax toxin proteins. Researchers have tried expressing and purifying PA in other microorganisms such as Bacillus subtilis, Baculovirus and E. coli. Purification of PA from Bacillus subtilis resulted in poor yields, required growth in rich media, and enormous amount of PA was degraded due to proteases secreted by the organism (L. W. J. Baillie et al, Lett. Appl. Microbial (1994) 19, 225-227). Baculovirus vectors expressed PA in insect cells; however, purification could not be possible due to low yields. Although PA has been expressed in E. coli, attempts to overproduce the protein were not successful (M. H. Vodkin et al, Cell, (1983) 34, 693-697). Researchers also purified PA by guiding the protein to the periplasmic spaces, however the yields of the purified PA were very low. All the known expression systems for Protective Antigen expression using E. coli, are inducible systems that require the use of IPTG, an expensive chemical.
U.S. Pat. No. 2,017,606 describes the preparation of anthrax antigen by growing Bacillus anthracis on a suitable culture medium and separating the bacilli from the culture medium.
U.S. Pat. No. 2,151,364 describes a method of producing an anthrax vaccine which comprises preparing the suspension of anthrax spores and adding to the suspension a sterile solution containing alum.
The drawbacks in the above US patents is that both of them use Bacillus anthracis cultures or spores. Bacillus anthracis is an infectious organism and cannot be handled without containment facilities. The levels of protective antigen expressed in Bacillus anthracis are very low. This kind of vaccine preparation is also contaminated with other toxic and non-toxic proteins from Bacillus anthracis resulting in a number of side-effects and reactogenicity.
The object of this invention is to create a constitutively expressing system for rapid, efficient, cost-effective and high-level production of anthrax PA from E. coli using Fed-Batch culture.
To achieve the said objective this invention provides a process for preparing anthrax protective antigen protein from E. coli using fed batch culture comprising:                transforming E. coli DH5α cells with the recombinant constitutive expression plasmid containing the PA gene to produce the recombinant DH5α cells expressing the PA protein,        growing said recombinant DH5α cells and testing the PA expression by lysis of said cells followed by denaturing gel electrophoresis and Western Blotting technique using PA antibodies,        fermenting said cells in a bio-reactor using:                    polyols, carbohydrates or organic acids as primary supplements in Luria Broth medium at 32-42° C.,            fed-batch culture technique, and            pH-DO stat method of sensing nutrient deprivation to produce high cell density culture expressing PA protein,                        harvesting said cells by centrifugation of said high cell density culture at 5000-10,000 rpm for 10-30 minutes,        solubilizing said high cell density culture cells by using 6-8 Molar Urea solution and stirring at ambient temperature for 1-2 hours,        separating said high cell density culture debris by centrifugation at 10,000-15,000 rpm for 30-60 minutes at 32-42° C. and collecting the supernatant containing urea denatured PA,        isolating said urea denatured PA from said supernatant and purifying it by Ni-NTA chromatography by gradual removal of urea while said PA is bound to the affinity column, and        eluting said purified renatured PA and storing protective antigen (PA) protein as frozen aliquots at −20 to −70° C. depending upon immediate or long term use.        
The said recombinant constitutive expression plasmid used expresses the PA protein as insoluble inclusion bodies in the E. coli strain DH5α cells.
The harvesting of said cells by centrifugation of said high cell density culture is carried out at 5000 rpm for 10 minutes.
The centrifugation of said high cell density culture debris is carried out at 10,000 rpm for 30 minutes for maximizing the harvesting of said cells.
The said polyol used as primary supplement in Luria Broth medium during fermentation is glycerol,
The said carbohydrates used as primary supplement in Luria Broth medium are glucose, galactose, maltose, fructose and lactose,
The said organic acid used as primary supplement in Luria Broth medium is malic acid.
By using polyol, carbohydrate or organic acid, as primary supplement in Luria Broth medium, the maximum cell density ranges between 10-14 optical density units in shake flask cultures.
The maximum cell density of the recombinant cells is achieved by Fed Batch cultures containing MgSO4.
The concentration of Luria Broth medium used in the feed is 5-25×.
The concentration of Luria Broth medium used in the feed is 25× in order to minimize the volume of feed added during fermentation.
The said plasmid is pQE series vector containing an E. coli recognizable phage promotor.
An anthrax antigen comprises of purified structurally, biologically and functionally active recombinant protective antigen (PA) protein of Bacillus anthracis expressed as a 6× histidine fusion protein in E. coli DH5α cells free from polysaccharides, dead bacteria, culture medium, water-soluble and insoluble by-products and suspended impurities.